New Zealand Patent Specification No. 237009 (PCT/CA90/00241 published as WO 91/01629) of British Columbia Research Corporation discloses the background to the clonal propagation of trees and other plants and discusses difficulties arising from precocious germination of embryos in vitro which does have an adverse effect on seed germination when germination is desired.
British Columbia Research Corporation in the aforementioned New Zealand Patent Specification No. 237009 (U.S. Pat. No. 5,183,757) discloses a process for a propagation of conifer somatic embryos which includes the steps of differentiating somatic embryos in contact with a growth medium, separating the somatic embryos from said medium, and germinating the somatic embryos, the process being characterised from prior art processes in that the embryos are matured on said medium and are partially dried prior to germination by exposing mature embryos separated from the said medium to an atmosphere having greater than 80% humidity. The species used for an example is spruce. The Specification discloses the establishment of such a humidity by providing in a closed area the presence of water either in the form of water itself or as part of an aqueous solution of a salt. The pregermination treatments included placing mature embryos in petri dishes on water-saturated kimpaks on petri dishes at room humidity or in 6 wells of a 12 well petri dish with the other 6 wells filled three quarters full with sterile water.
We have found that when this method is applied to pine, all embryos elongated and some became partially green even though stored in the dark at all times (unpublished). This rendered them unsuitable for germination as they had already partially germinated precociously.
U.S. Pat. No. 5,238,835 granted Aug. 24, 1993 (University of Guelph) provides additional background as to the use of embryogenesis in the creation of artificial "seeds". There is reference therein to U.S. Pat. No. 4,615,141 (Janick and Kitto) concerning methods for enhancing desiccation tolerance in somatic embryos.
U.S. Pat. No. 5,238,835 itself discloses in relation to Alfalfa and Brassica a pregermination treatment comprising the application of an environmental stress to cause the embryos to synthesise abscisic acid in sufficient quantity to cause expression of desiccation tolerance in the embryos. The application of environmental stress is stated as comprising one or more of
(i) slow gradual desiccation
(ii) low temperature
(iii) nutrient starvation
(iv) heat stress, and
(v) osmotic stress.
The benefits stated are a) prolonged storage in a dormant state and b) enhanced vigour of the resultant seedling. It is stated that when dry the metabolism of the embryo is arrested and thus the utilisation of valuable storage reserves and the precocious growth of the seedling is prevented. It is stated as anticipated that tissue prepared and dried as described in the procedure could be stored in a similar way and for similar periods of time compared to "true" seeds. It is additionally stated that there is enhanced vigour of the seedling from a dry embryo compared to that produced by an embryo which follows a developmental path analogous to precocious germination.
U.S. Pat. No. 5,238,835 indicates that once tolerance has been induced the embryo can be air dried to moisture contents less than 20% water or equivalent to those observed in true seeds. Such dried embryos are stated as being capable of being stored in cool dry conditions such as those used for storing true seed.
There is an indication that the procedure necessarily is of two parts.
(i) the subjection of the embryos to abscisic acid, and
(ii) thereafter, (eg. after about 5 to 14 days of the exposure to abscisic acid) the adoption of a "fast" or "slow" drying procedure.
The abscisic acid treatment (i) in the preferred form is as a result of the aforementioned subjection to environmental stress causing the embryos to synthesize the abscisic acid required by the step (i).
It is stated that "fast drying" of step (ii) is achieved either by air drying or by drying in a low humidity (45% relative humidity) chamber. It is also stated that "slow drying" of step (ii) is achieved by placing embryos in a series of desiccators each with a controlled humidity for a total of 6 days. For the 1st day of drying embryos are kept at 97% humidity and are transferred daily to chambers with 87%, 75.5%, 62.5%, 50.5% and finally to 43% relative humidity. This invention does not require any "drying" of embryos.
Earlier U.S. Pat. Nos. 5,041,382, 4,957,866, 5,036,007, 5,236,841 and PCT WO 93/1166 refer to the pretreatment of embryos with osmotic agents (such as Polyethyleneglycol PEG). These are claimed to enchance the quality of somatic embryos at different stages including the mature stage and to allow efficient desiccation of embryos and subsequent germination. We have found these and other osmotic treatments unnecessary for the purpose of this invention and the somatic embryogenesis process in general, and use of them inhibits the development of embryos, including the maturation stage (data not shown). The tissue grows into an undesirable form (fluffy-like), and formation of embryos at any stage is inhibited in experiments to date.
Other authors also do not use a desiccation protocol (Zelu, M. A., Bastien, C., Klimaszewska, K. and Chacest, P (1994) An improved method for somatic plantlet production in hybrid larch: Part 2, Control of germination and plantlet development. Plant Cell, Tissue and Organ Culture 36(1):117-127).
PCT WO 93/1166 also claims the preferred moisture content of conifer somatic embryos as ranging between 10-55% accomplished with the use of osmotic agents (eg; PEG) in the medium, in order to store somatic embryos for long periods of time. The present invention provides an alternative to the methods of PCT W093/1166, ie; we require the addition of osmotic agents and we avoid the requirement for lower moisture content in the 10-55% range.
New Zealand Forest Research Institute (FRI) patents [South African Patent No. 93/4807] states that a pre-germination media prepared with a gelling agent or no pre-germination between maturation of embryos on ABA-containing media (4--see FIG. 4) and germination of embryos (6--see FIG. 4) are the preferred methods. When these methods were tested 81.3% of embryos stayed white or became partially green after 4 weeks of germination, and only 18.7% germinated. In this respect please see Table 1.
TABLE 1 Germination of radiata pine embryos using protocols in FRI patents Percentage Low Quality High Quality Germination Clone No. Total Embryos Embryos Embryos after 4 weeks A93 55 7 33 27.3 A93-17 24 2 15 29.2 C93-4 43 2 19 51.2 D93-199 236 27 174 14.8 E93-2 78 18 45 19.2 I93-22 217 55 134 12.9 Total 653 222 420 18.7
Other prior art refers to storage of embryogenic tissue at the earlier maintenance stage of embryogenesis @ 5, 10, 15, 25.degree. C. (2--see FIG. 4) by Y. S. Park, S. W. Pond & J. M. Bonga (1994): Somatic embryogenesis in white spruce (Picea glauca): genetic control of somatic embryos exposed to storage, maturation treatments, germination and cryopreservation. Theor. Appl. Genet. 89:742-750.